Hi everybody,
I have a linear ligation product composed of 2 pieces (about 3kb in size). When I ran the ligation reaction on the gel, there were some unspecific bands as well as a faint band of around 3kb which seems to be the ligated band of interest. So I tried to amplify the 3kb band using Forward primer of the 1st fragment and Reverse primer of the 2nd one. Unfortunately I get only smear after amplification. Even changing annealing temperature (from 60-70) didn't affect positively.
I use high fidelity Phusion polymerase which recommend higher Ta (Tm+3), however it doesn't work. Also I tried different Mg concentration, but without promising results.

Tm(F)= 68.8
Tm(R)= 67.3

the questions are;
1. How can I ligate two or more linear fragments much more effectively and with higher yield for band of interest?
2. How can I amplify the ligated band of interest which is faint?

Should it seem possible to you kindly help me to find the solution.

many thanks

-skad-

You'll either need to isolate the faint 3 kb band from the ligation mixture by gel purification and use that as a PCR template, or clone the fragments into a vector and do colony PCR to find clones that contain the correct fragment.

-HomeBrew-

skad on Sat Dec 11 11:38:28 2010 said:

the questions are;
1. How can I ligate two or more linear fragments much more effectively and with higher yield for band of interest?
2. How can I amplify the ligated band of interest which is faint?

Should it seem possible to you kindly help me to find the solution.

many thanks

1. What you need is a termination fragment to terminate the ligation reaction. ie you need a vector fragment to ligate to the ends of the two inserts and form a circular plasmids (that you can then later amplify). Without a termination fragment (plasmid vector), the ligation reaction will continue and continue. It should be noted that a ligation between insert A and insert B can produce three initial product, insertA-insertA, insertB-insertB and insertA-insertB. All three initial product will amplify with PCR using primers for A and B Thus PCR is of no use here.

2. Ligate the two inserts into a plasmid (multiway ligation) and transform said plasmid into cells. screen for a colony containing the two inserts. Grow said colony and extract the plasmid. Lot of product.

-perneseblue-

Hi Veteran, tnx for your reply,
I already did ur suggestion, but the result was smear and when I tried different strategies to optimize PCR and remove smears, there were not any band and just some very short unspecific bands.

-skad-

Tnx "Unlimited ligation works!"
sorry I didn't get what you recommend about termination fragment.
but I think among the initial products when forward primer for A fragment and reverse primer for B fragment is used in PCR, only insertA-insertB will be amplified. because both of the insertA-insertA and insertB-insertB doesn't have any priming site for second primer. Am I right?